FIG.5.

Pak5 contains a functional NLS. (A) Schematic representation of primary structure of Pak5 showing the various protein domains, in particular the NLS. (B) CHO cells were transfected with GFP-wt-Pak5 Δ5-10, GFP-mut-Pak5 Δ5-10, or GFP-mut-Pak5 as a control. Cells were stained with MitoTracker, and cellular localization was visualized by confocal microscopy. Bar = 20 μm. (C) Stable cell lines expressing Myc-tagged wt-Pak5 Δ5-10 or mut-Pak5 Δ5-10 were established. (Left panel) Immunoblot showing expression of both proteins. (Right panels) Cellular fractionation was performed 48 h after transfections, as described in Materials and Methods, to assess Pak5 distribution to various cellular compartments. Equal amounts of proteins from the cytosolic (S100), light microsome (L), and mitochondrial (M) fractions were loaded on a gel, and immunoblotting was performed using anti-Myc antibodies. Anti-COX IV (for mitochondria) and anticalnexin (for light membranes) antibodies were used as controls. (D) Nuclear extraction was performed as described in Materials and Methods on the stable cell lines described above. Anti-PCNA antibodies were used as a loading control. (E) Stable CHO cells expressing Myc-wt-Pak5 Δ5-10 or Myc-mut-Pak5 Δ5-10 were treated for 12 h with CPT (10 μM). Cells were then collected and stained with annexin V and propidium iodide. Apoptosis was assessed by flow cytometry. The percentages of total cells positive for annexin V are shown in the graph and represent the averages of three experiments.